The present invention, certain aspects of which are published in Virus Research (1999), 63:75-83 and J. Virol. (1999), 73:5056-5063 (which are herein incorporated by reference), relates to apoptosis in aquatic organisms induced by aquabimavirus, preferably infectious pancreatic necrosis virus (IPNV). It also relates to methods to regulate apoptosis in aquatic cells by controlling the expression of Mcl-1 gene and pre-treating the aquatic cells with drugs which block the viral replication. It further relates to a method for monitoring the progress of apoptosis using EGFP (a variant type of green fluorescent protein [GFP]) as a probe.
Infectious pancreatic necrosis virus (IPNV) is an economically important fish pathogen. IPNV belongs to a group of viruses known as Birnaviridae (Brown (1986), Intervirology, 25:141-143). Other members of Bimaviridae include infectious bursal disease virus (IBDV) of fowl and Drosophila X virus. IPNV was discovered to be associated with a highly contagious disease of susceptible hatchery-reared trout and salmonoids. As the name indicates, the infection among trout produces marked pancreatic necrosis, but histopathological changes sometimes also occur in adjacent adipose tissue, in renal hematopoietic tissue, in the gut, and in the liver (Wolf et al.(1960), Proc. Soc. Exp. Biol. Med., 104:105-108). Histopathological changes can also occur in renal excretory and hematopoietic tissues, as first reported by Yasutake et al. (1965), Ann. N. Y. Acad. Sci., 126:520-530. Although renal damage is consistent with the high titer of virus typically found in kidneys, at least in carrier fish, focal degeneration of liver parenchymal cells in yearling Atlantic salmon which had been previously inoculated with IPNV was also noted (Swanson and Gillespie (1979), J. Fish. Res. Board Can., 36:587-591).
IPNV shows a high degree of antigenic heterogeneity. Three different stereotypes, Ab, Sp and VR299 (MacDonald and Gower (1981), Virology, 114:187-195; Okamoto et al. (1983), Eur. J. Fish Dis., 6:19-25) and ten subgroups (Heppell et al. (1992), J. Gen. Virol., 73:2863-2870) have been identified. IPNV is the etiological agent of a contagious, high mortality disease of young, hatchery-reared salmonids (Wolf et al. (1960), supra) and other non-salmonid fishes (Adair and Ferguson (1981), J. Fish Dis., 4:69-76). IPNV is a double-stranded RNA virus with four virion proteins (MacDonald and Dobos (1981), Virology, 114:414-422).
Birnaviruses possess a bisegmented (A and B), double-stranded RNA genome contained within a medium-sized, unenveloped, icosahedral capsid. Birnavirus gene expression involves the production of four unrelated major genes that undergo various posttranslational cleavage to generate three to five structural proteins (Dobos, P. 1995. Annu. Rew. Fish Dis. 5, 25-54). The largest protein (90 kDa), VP1, is encoded by the smaller segment B genome, and the larger genome segment A encodes VP2 (42 kDa), VP4 (28 kDa) and VP3 (32 kDa). Genome segment A contains an additional small open reading frame (ORF) which overlaps the amino terminal end of the polyprotein from the reading frame (Duncan, R., Nagy, E., Krell, P. J. and Dobos, P. 1987. J. Virol. 61, 3655-3664). This small ORF encodes a 17 kDa arginine-rich minor polypeptide, VP5, which is produced in small quantities and is synthesized during the early replication cycle (Magyar, G. and Dobos, P. 1994. Virology 204, 580-589).
There are two major morphologically and biochemically distinct modes of death in eukaryotic cells: apoptosis and necrosis (Duvall and Wyllie (1986), Immunol. Today, 7:115-119). Apoptosis is a physiological process involved in normal tissue turnover that occurs during embryogenesis, aging, and tumor regression, but pathological stimuli, such as viral infections (Gougeon and Montagnier (1993), Science, 260:1269-1270), can also be triggering factors. Typically, apoptotic cell death is characterized by nuclear condensation, endonucleolytic degradation of DNA at nucleosomal intervals (xe2x80x9cladderingxe2x80x9d), and plasma membrane blebbing (Wyllie et al. (1980), Int. Rev. Cytol., 68:251-306). Necrosis is a pathological reaction that occurs in response to perturbations in the cellular environment such as complement attack, severe hypoxia, or hyperthermia. These stimuli increase the permeability of the plasma membrane, resulting in irreversible swelling of the cells (Wyllie et al,(1980), supra).
Apoptosis is important during embryonic development, metamorphosis, tissue renewal, hormone-induced tissue atrophy and many pathological conditions. In multi-cellular organisms, apoptosis ensures the elimination of superfluous cells including those that are generated in excess, have already completed their specific functions or are harmful to the whole organism. In reproductive tissues, massive cell death occurs under the control of hormonal signals. A growing body of evidence suggests that the intracellular xe2x80x9cdeath programxe2x80x9d activated during apoptosis is similar in different cell types and conserved during evolution. (Hengartner and Horvitz (1994), Cell, 76:1107-1114). In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer.
Apoptosis involves two essential steps. The xe2x80x9cdecisionxe2x80x9d step is controlled by the Bcl-2 family of proteins which consists of different anti- and pro-apoptotic members. The xe2x80x9cexecutionxe2x80x9d phase of apoptosis is mediated by the activation of caspases and cysteine proteases that induce cell death via the proteolytic cleavage of substrates vital for cellular homeostasis.
Bcl-2 protein is a 25 kD integral membrane protein of the mitochondria. Bcl-2 protein extends survival in many different cell types by inhibiting apoptosis elicited by a variety of death-inducing stimuli (Korsmeyer (1992), Blood, 80:879-886). Overexpression of bcl-2 has been related to hyperplasia, autoimmunity and resistance to apoptosis (Fang et al. (1994), J. Immunol., 153:4388-4398). Bcl-2 contains a family of related genes, which includes, but is not limited to, A1, mcl-1, bcl-w, bax, bad, bak and bcl-x. A1, mcl-1, bcl-2 and bcl-x1 (long form of bcl-x) are presently known to confer protection against apoptosis and are referred to herein as xe2x80x9canti-apoptotic bcl-2 related proteinsxe2x80x9d. In contrast, bax, bad, bak and bcl-xs (short form of bcl-x) are presently known to promote cell death by inhibiting this protective effect.
Mcl-1 is one of the members of the Bcl-2 family. Like Bcl-2, Mcl-1 heterodimerizes with Bax, an accelerator of apoptosis in the Bcl-2 family, and neutralizes the cytotoxicity induced by Bax in yeast (Bodrug et al. (1996), Death Differ., 2:173-182). Mcl-1 is also able to protect Chinese hamster ovary cells from apoptosis induced by c-myc overexpression (Reynolds et al (1994), Cancer Res., 54:6348-6352). This protein was discovered as a novel gene induced early in the induction of differentiation of a human myeloid leukemia cell line (Kozopas et al. (1993), Proc. Natl. Acad. Sci. USA, 90:3516-3520). Expression of Mcl-1 mRNA was rapidly up-regulated with phorbol ester in those cells followed by a rapid degradation, consistent with the presence of a mRNA destabilization sequence in its 3xe2x80x2-untranslated region. The half-life of the Mcl-1 protein is short (Yang et al. (1995), J. Cell Biol., 128:1173-1184), which has been ascribed to the presence of two PEST (proline, glutamic acid, serine, threonine) motifs. Therefore, Mcl-1 is suggested as a rapidly inducible, short-term effector of cell viability (Yang et al. (1996), J. Cell. Physiol., 166:523-536). Recently, Hong et al. (Virology, (1998), 250:76-84) reported that an El-S of IPNV Ab strain induced apoptosis in CHSE-214 cells. Hong et al.""s publication is herein incorporated by reference. In Hong et al.""s report, four kinds of detecting methods were used to determine whether apoptosis is involved in fish embryonic cell death after IPNV infection: (1) assay with terminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection; (2) assay for procoagulant activity; (3) assay for DNA ladders; and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells.
The results show that apoptosis precedes any detectable necrotic change in CHSE-214 cells, suggesting that apoptosis characterizes the onset of pathology in host cells and is followed by necrotic processes. Hong et al.""s report is important because previously, IPNV infection is only viewed as caused by a necrotic process. However, Hong et al.""s report did not provide any insights which delineate the apoptotic process from necrosis.
In the present invention, an investigation of apoptosis is carried out by using CHSE-214 cells infected with IPNV as a model. The investigation is conducted by transfecting the cells with a pEGFP vector which enables the cells to express EGFP (a variant type of GFP [green fluorescent protein]). Based on the special characteristics of EGFP which can fluorescence 35 times more intense than the wild-type GFP, the morphological changes during apoptosis are monitored, which show that IPNV causes CHSE-214-EGFP cells to undergo apoptosis, then a nontypical apoptosis, and finally, postapoptotic necrosis in cells. The discovery of the nontypical apoptosis stage before necrosis takes place is one of the novel findings in the present invention.
The present invention also provides studies of apoptosis via an Mcl-1 dependent pathway. The results of the present invention indicate that the occurrence of apoptosis is due to down regulation of the Mcl-1 gene caused by viral infection. In addition, various drugs or chemicals are tested for their capacity of preventing the down-regulation of Mcl-1 protein expression by viral infection. The results show that by blocking the down regulation of the Mcl-1 gene, the cell death caused by IPNV infection is effectively prevented.
The present invention is important because it not only provides a model for studies of apoptosis but also provides a means for preventing or containing widespread of IPNV infection in aquatic organisms.
The present invention provides an aquatic apoptotic cell which can be used as a model for studying morphological changes during apoptosis. The aquatic apoptotic cell is induced by infecting an aquatic cell with an aquabirnavirus. The preferred aquatic cell is a fish cell. The preferred fish cell includes, but is not limited to, salmon, trout, grouper, and eel cells. The most preferred fish cell is Chinook salmon embryo cell (CHSE-214). The preferred aquabimavirus is an infectious pancreatic necrosis virus (IPNV). The preferred IPNV is El-S of IPNV Ab strain which is isolated from Japanese eel in Taiwan (Wu et al. (1987), Bull. Inst. Zool. Acad. Sinica, 26:201-214). The infectious period is preferred not to exceed 8 hours.
The morphological changes of the aquatic apoptotic cell during apoptosis can be monitored by fluorescence using EGFP as a probe. EGFP is introduced into the aquatic cell by transfecting the cell with a pEGFP-N1 vector. By using EGFP, a nontypical apoptotic process is discovered which occurs after a typical apoptosis and before the necrosis process. This nontypical apoptosis features, including highly condensed membrane blebbing, occurs during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and are finally pinched off from the cell membrane. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell. The results also demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.
The present invention also provides agents for inducing or preventing/rescuing apoptosis. The first agent for inducing apoptosis is IPNV itself. The second agent for inducing apoptosis is VP3, a 32-kDa protein derived from the IPNV segment A. The nucleotide sequence of VP3 is publicly available, as accession number AF291752 in NCBI gene data bank; website address: www.ncbi.nlm.nih.gov. The VP3 gene can be converted into cDNA by RT-PCT and inserted into a plasmid to transfect a host organism or a cell line.
The first agent for preventing/rescuing apoptosis caused by IPNV or VP3 is an antisense RNA of VP3, which can be transfected into a host or a cell line. The second agent for preventing/rescuing apoptosis caused by IPNV or VP3 is a zfmcl-1a gene, which can be inserted into a plasmid (such as pEGFP-zfMcl-1a) for transfection into a host or a cell line.
The present invention also includes two methods for detecting apoptosis. The first method provides a means to visualize morphological changes during apoptosis. The method contains the following steps: (1) transfecting the aquatic cells with a pEGFP-N1 vector; (2) infecting the aquatic cell with an aquabimavirus; and (3) monitoring the morphological changes by a microscopic technique. The pEGFP-N1 vector is driven by an immediate-early promoter of human cytomegalovirus. The coding region contains the EGFP gene, which contains a chromophore mutation which produces fluorescence 35 times more intense than that of wide-type GFP (green fluorescence protein). GFP is a revolutionary molecule which can be used to monitor gene expression and fusion protein localization in vivo or in situ and in real time. GFP is from the jellyfish Aequorea victoria. The transfected cells can be screened by G418. The preferred aquabimavirus is IPNV. The preferred aquatic cell is CHSE-214. The microscopic technique includes, but is not limited to, light microscopy, fluorescence microscopy, scanning electron microscopy, and immuno-electron microscopy.
The second method provides quantitative measurements of apoptosis. The method contains the following steps: (1) transfecting an aquatic cell with a pEGFP-N1 vector; (2) infecting the aquatic cell with an aquabimavirus; and (3) measuing EGFP in the aquatic cell and in the culture medium. EGFP is measured based upon the fluorescence intensity. The pEGFP-N1 transfected aquatic cell produces EGFP which can be evaluated by a Fluorolite 1000 (DYNEX).
Furthermore, the present invention provides methods for inducing or preventing apoptosis in vivo or in vitro. The first method for inducing apoptosis includes in vivo infection of aquatic organisms with an aquabimavirus. The preferred aquabimavirus is IPNV. The preferred IPNV is El-S of IPNV Ab strain which is isolated from Japanese eel in Taiwan (Wu et al. (1987), supra). The preferred aquatic organisms are embryos or hatchery-reared juvenile salmonids and nonsalmonid fish. Salmonid fish include, but are limited to, pacific salmon in general (Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fish includes carp, perch, pike, eels and char. Certain mollusks and crustaceans can also be infected with IPNV.
This method also applies to in vitro transfection of IPNV into fish cell lines, such as CHSE-214 (an embryo cell line from salmon). The preferred temperature for infected cultures is at 18xc2x0 C. The induction of apoptosis by aquabimavirus can be prevented by pretreatment of cyclohexamide (protein synthesis inhibitor), genistein (tyrosine kinase inhibitor), and EDTA (cation chelator) prior to viral infection.
The second method for inducing apoptosis includes transfection of a VP3 gene-containing plasmid into a host, which may be an aquatic/vetebrate organism or cell line, to overexpress VP3 in the host. VP3 is a 32-kDa protein derived from the IPNV segment. The nucleotide sequence of VP3 is publicly available, as accession number AF 291752 in NCBI gene data bank; website address: www.ncbi.nlm.nih.gov.
The VP3 gene of IPNV can be converted into DNA by RT-PCR and constructed into a plasmid containing EGFP to form a pEGFP-VP3 by techniques which are known to persons with ordinary skill in the art. The preferred aquatic host includes salmonids and nonsalmonid fish. Salmonid fish include, but are limited to, pacific salmon in general (Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fish includes carp, perch, pike, eels, zebrafish and char. The preferred vetebrate host includes rat, hamster and human. The preferred aquatic or vertebrate organism is embryo. The pEGFP-VP3 can transfect with liposome to cell lines or a vertebrate embryo by microinjection by commonly known methods. The preferred cell lines for induction of apoptosis by VP3 include CHSE214 (an embryo cell line from salmon), NIH3T3 (mice fibroblast), CHO (Chinese hamster ovary cell), and Hepa 3b and Hepa G2 cells (human liver tumor cells).
The third method for inducing apoptosis include down regulating Mcl-1 gene expression. The down regulation of Mcl-1 gene expression correlates to IPNV replication. The preferred aquatic organisms are fish, in particular fish cells derived from salmon, trout, grouper, and eel. The down regulation of Mcl-1 gene expression can be blocked by drugs such as cyclohexamide (protein synthesis inhibitor), genistein (tyrosine kinase inhibitor), and EDTA (cation chelator). These drugs help to maintain Mcl-1 expression level and block the induction of DNA internucleosomal cleavage (i.e., blocking the intense DNA laddering pattern), so as to rescue or delay the apoptotic cell death process.
The first method for preventing or rescuing apoptosis by IPNV infection or VP3 transfection requires transfecting an antisense RNA of VP3 into a host, which may be an aquatic/vertebrate organism or cell line. The preferred aquatic host includes salmonids and nonsalmonid fish. Salmonid fish include, but are limited to, pacific salmon in general (Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fish includes carp, perch, pike, eels, zebrafish and char. The preferred vetebrate host includes rat, hamster and human. The preferred aquatic or vertebrate organism is embryo. The pEGFP-VP3 can transfect an aquatic or a vertebrate embryo by microinjection by commonly known methods. The preferred cell lines include CHSE-214 (an embryo cell line from salmon), NIH3T3 (rat fibroblast), CHO (hamster embryoic cells), and Hepa 3b and Hepa G2 cells (human liver tumor cells).
The second method for preventing or rescuing apoptosis from IPNV infection requires transfecting a zfMcl-1a into a host, which may be an aquatic/vertebrate organism or cell line. The nucleotide sequence of zfMcl-1a is publicly available, which is ZfMcl-1a accession number AF 231016 as published in NCBI gene data bank; website address: www.ncbi.nlm.nih.gov. The zfMcl-1a can be transfected into the host by microinjecting a zfMcl-1a-containing plasmid such as pEGFP-zfMcl-1a or pEGFP-C1. The preferred aquatic host includes salmonids and nonsahnonid fish. Salmonid fish include, but are limited to, pacific salmon in general (Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fish includes carp, perch, pike, eels, zebrafish and char. The preferred vertebrate host includes rat, hamster and human. The preferred aquatic or vertebrate organism is embryo. The pEGFP-VP3 can transfected an aquatic or a vertebrate embryo by microinjection by commonly known methods. The preferred cell lines for preventing or rescuing apoptosis include CHSE-214 (an embryo cell line from salmon), NIH3T3 (mice fibroblast), CHO (Chinese hamster ovary cell), and Hepa 3b and Hepa G2 cells (human liver tumor cells).